Vasopermeability-enhancing conjugates

ABSTRACT

Liposomal conjugates having a clinically useful delivery vehicle linked to a biologically active species which acts to increase vascular permeability and expand blood volume at or in proximity to the tumor site are disclosed. The vehicle-linked species may be, for example, a vasoactive agent, a substance that recruits or amplifies a vasoactive species, a drug, or a pharmaceutical compound. Suitable biological species comprises peptides, lipids, carbohydrates, or their derivatives. Chemical or recombinant DNA methods suitable for linking the species to the vehicles are indicated. A therapy is disclosed which comprises administering the vasoactive conjugate and delivering a diagnostic agent or a therapeutic agent at an optimal time thereafter, when tumor vasculature is maximally affected.

RELATION TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/916,883, now U.S. Pat. No. 6,524,823, filed on Jul. 27, 2001, whichis a continuation of U.S. patent application Ser. No. 09/382,359, nowU.S. Pat. No. 6,274,343, filed on Aug. 24, 1999, which is a continuationof U.S. patent application Ser. No. 08/419,645, now U.S. Pat. No.6,007,817, filed on Apr. 10, 1995, which is a continuation of U.S.patent application Ser. No. 08/127,988, filed on Sep. 27, 1993,abandoned, which is a continuation of U.S. patent application Ser. No.07/964,517, filed on Oct. 21, 1992, abandoned, which is a continuationof U.S. patent application Ser. No. 07/417,782, filed on Oct. 4, 1989,abandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 07/255,513, filed on Oct. 11, 1988, abandoned. Each of theabove mentioned patents is incorporated by reference herein, in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the use of immunological agents and otheragents with unique specificities in vivo, and particularly, to means forenhancing the penetration and binding of monoclonal antibodies and othermacromolecules used for the diagnosis and therapy of human disease.

2. Description of the Related Art

The use of tumor-specific monoclonal antibodies (mAbs) has been activelyinvestigated in therapy directed at several different types of humancancers (Levy and Miller, Fed. Proc. 42: 2650-2656 (1983)), and to date,a number of clinical trials have been reported. Both phase I and IIlevels of clinical trials have convincingly demonstrated the safety ofthese agents, even at high dose levels; but they also indicate thatmonoclonal antibodies (“mAbs”) have not been as effective in vivo aspredicted.

The effectiveness of antibodies to tumor-associated antigens in thetherapy of cancer depends on the ability of antibodies to destroy theirtarget cells by either direct cytotoxicity or complement-mediatedcellular lysis. Complement-mediated lysis is triggered when the Clqcomponent of the classical complement pathway binds to the Fc portion ofantibodies bound to the surface of tumor cells, leading to the formationof the membrane attack complex. Tumor-bound antibodies can also recruitthe natural defenses of the host by interacting with effector cellswhich themselves lyse the target. However, despite their multiplecytotoxic capacity, the actual experimental use of mAbs alone ascytotoxic agents has been unsatisfactory. The trials have resulted insome remissions, but in general most patients have had only minorresponses which are often transient in nature (Foon et al., Blood 64:1085-1093 (1984); Sears et al., Cancer Res. 45: 5910-5913 (1985)).

Investigators have attempted to improve the therapeutic effectiveness ofmonoclonal antibodies by supplementing the cytotoxicity of the antibodymolecule itself with cytotoxic radionuclides, toxins, and drugs attachedthereto (DeNardo, S. et al., Nucl. Med. Biol.13: 303-310 (1986);Hurwitz, E. et al., Cancer Research 35: 1175-1181 (1975); Ghose, T. etal., J. Natl. Cancer Inst. 58: 845-852 (1977)).

Attempts to improve the tumoricidal capacity of mAbs have also includedattaching biological response modifiers to provide antibody conjugatesthat would also provoke a local natural immune response at the antibodybinding site.

One example of this use of biological response modifiers are conjugatesof antibody and cobra venom factor (CVF). CVF is a glycoprotein, havingthe properties of the C3b, C3/C5 convertase of the alternative pathwayof complement. However, CVF, unlike its native analog, is notinactivated by complement control proteins. The presence of CVF oncell-bound antibody initiates assembly of the membrane attack complexand thereby cell death. (Vogel, C. and Muller-Eberhard, H., Proc. Natl.Acad. Sci., USA. 78(12): 7707-7711; Vogel, C. et al., “Hematology andBlood Transfusion”, in Modern Trends in Human Leukemia VI, 29: 514-517(1985) Berlin Neth, et al.).

Another example is the use of immunoconjugates comprising monoclonalantibody and interferon, in which interferon enhances target cell lysisby activation of preexisting cellular immune mechanisms, includingnatural killer (NK) cells. (Flannery, G. et al., Eur. J. Cancer Clin.Oncol. 20(6): 791-798 (1984).)

Other investigators have studied the effects of immunoconjugatescomprising a chemotactic agent, formyl-methionyl-leucyl-phenylalanine(fMLP) which acts to increase monocyte/macrophage concentrations at thesite of tumor-bound antibody. (Obrist, R., Sandberg, A., CellularImmunology 81: 169-174 (1983); Obrist, R., et al., Bent 53: 251 (1986)).None of these efforts, however, have substantially improved the clinicaleffectiveness of antibody tumor therapy.

Studies show that this lack of clinical effectiveness is due in largepart to the delivery of insufficient quantities of mAbs to the tumorsite. Examination of tumor tissue by histochemical methods before andafter therapy indicated that even at high dose levels, there is only apartial saturation of tumor by antibody. (Lowder, et al., Blood 69:199-210 (1987)). Quantitative dosimetry studies using radiolabeledantibody preparations have revealed that only a very low percent oftotal dose actually binds to the tumor (0.05-0.2%) despite the highspecificity of the antibodies used or the achievement of hightumor:organ ratios. Studies with tumor-specific monoclonal antibodiesindicate that even with good tumor to blood distribution ratios, theabsolute amount of radiolabeled mabs detected per gram of tumors isabout 0.015% of the total injected dose. (Epenetos et al., CancerResearch 46: 3183-3191 (1986)).

Within the body, the primary mode of communication and delivery ofsubstances is via the circulatory system. In general, the circulatorysystem comprises the blood vascular system and the lymphatic system. Theblood vascular system, which distributes nutritive materials, oxygen,hormones and other substances to all parts of the body while removingthe products of cellular metabolism, includes the heart and a series oftubular vessels: the arteries, veins, and capillaries. The arteries,which by branching constantly increase in number and decrease incaliber, conduct blood from the heart to the capillary bed. Thecapillaries, where the interchange of elements between the blood and theother tissues takes place, form a meshwork of anastomosing tubules.Veins, in turn, return blood from the capillaries to the heart.

The capillaries are typically comprised of simple endothelial cells thatconnect the arterial and venous sides of the circulatory system. Meshesof the capillary network are present throughout the body, varying insize and in shape in different tissues and organs. The intensity ofmetabolism in a region generally determines the closeness of the mesh.Therefore, there is a close network in the lungs, liver, kidneys, mucousmembranes, glands, and skeletal muscle, as well as in the grey matter onthe brain. The network has a large mesh and is sparse in tissues such astendons, nerves, smooth muscle, and serous membranes.

The ability to transfer substances through the wall of capillaries isreferred to as permeability. Permeability varies regionally and, underchanged conditions, locally.

In general, it is agreed that tumors must induce a new blood supply ifthey are to grow beyond a diameter of a few millimeters, and a greatdeal of attention has been focused on the mechanisms by which tumorsinduce angiogenesis. (For example, see Folkman, J., Adv. Cancer Res. 43:175-263 (1985).) Significant attention has also been devoted to theanatomy and physiology of the new blood vessels that come to supplytumors. (Id.)

It is generally agreed that tumor vessels are anatomically heterogeneousstructures. Often, they consist of relatively undifferentiated channels,lined by a simple endothelium and with fewer pericytes and smooth musclecells than would be expected of comparably sized vessels in normaltissues. The functional properties of tumor vessels have been morecontroversial; tumor vessels have been reported to be either more orless responsive to vasoactive mediators than normal vessels. (See, e.g.,Hori, K., et al., J. Natl. Cancer Inst. 74: 453-459 (1985).) Oneproperty of tumor vessels on which most investigators agree, however, isthat, relative to normal vessels, tumor vessels are hyperpermeable tocirculating macromolecules. This observation demands explanation becauseof its obvious relevance to an understanding of the localization ofmonoclonal antibodies and tumoricidal drugs in solid tumors. (See, e.g.,Dvorak, et al., Am. J. Pathol. 133: 95-109 (1988).) Whereas smallmolecules pass freely through normal capillaries and other vessels withintact interendothelial cell junctions, the permeability of the normalvasculature to macromolecules is tightly regulated. Normally,macromolecules are largely retained within the circulation and the smallamounts that do escape are thought to do so by means of vesiculartransport or by the formation of transient transcytoplasmic channelsacross endothelial cells. (See, e.g., Milici, H. A., et al., J. CellBiol. 105: 2603-2612 (1987).) In inflammation, however, the escape ofmacromolecules is greatly increased; agonists such as histamine provokea contraction of post-capillary endothelial cells, resulting in theformation of interendothelial cell gaps through which macromolecules andeven particulates may escape. Regardless of whether or not tumorvasculature is “leaky”, however, we must reiterate that many studiesindicate that insufficient quantities of monoclonal antibodies are beingdelivered to the tumor site.

We believe that the reasons for the inadequate perfusion of tumors byblood are largely anatomical. Tumor cells grow radially from a centralcore of cells, rapidly outgrowing their blood supply, and leaving anecrotic, hypoxic core. In this instance, the distance from tumor cellsto the nearest capillary is about 100 to 150 μm, a distance great enoughto produce significant hypoxia and a perfusion deficit. These hypoxiccells show resistance to radiation and in addition, are inaccessible toinjected drugs or antibodies. (Kaelin, W. et al., Cancer Research 44:896-899 (1984); Thomlinson, P. and Gray, L., Br. J. Cancer 9: 539-549(1955)).

Limitations on mAb tumor therapy therefore appear to arise primarilyfrom transport-related factors such as the ability of the mAb topenetrate into the tumor and to localize and persist at the tumor site.The inefficient delivery and binding of mAbs to tumor cells and thelimitations it places on their clinical effectiveness is a majorobstacle to their use for diagnosis and therapy. The use of potentiatingagents, such as radioactive species, chemotherapeutic agents and toxicdrugs attached to the mAbs does not overcome this obstacle. Indeed,unless the mAbs are well concentrated at the tumor site, these attachedpotentiating agents carry the risk of increased damage to normaltissues.

Studies show that uptake of mAbs by tumor tissue correlates well withvascular permeability and blood flow (Sands et al., Cancer Res. 48:188-193, (1988)). A similar study indicates that administration of avasoactive agent may under some circumstances increase the perfusion oftumor relative to other tissues and increase tumor uptake andconcentration of radiopharmaceuticals. (Bomber, P. et al., J. Nucl. Med.27: 243-245 (1986)).

It is therefore an object of the invention to provide a specificallytargeted agent which can be used to increase vascular permeability andexpand tumor blood volume prior to the administration of tumoricidalimmunotherapy or chemotherapy so as to make that therapy more effective.

The same considerations of inefficient delivery also apply to the use ofspecifically targeted agents used in vivo for diagnostic imagingpurposes. An increased amount of an immunodiagnostic agent delivered tothe tumor site will improve the accuracy of the diagnostic procedure andallow a more efficient use of diagnostic agents, and a greater degree ofsafety to the patient in cases where the immunodiagnostic agent, such asradioisotope-labeled antibodies, carries some risk. It is therefore anobject of the invention to provide agents which will similarly enhancethe delivery of immunodiagnostic agents to a tumor by the specifictargeting of vasoactive agents to the site prior to the immunodiagnosticprocedure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of Lym/IL-2 immunoconjugate onradiolabeled Lym-1 F(ab′)₂ mice uptake in Raji-bearing nude mice.

FIG. 2 illustrates the effects of coinjection of IL-2 and Lym-1/IL-2vasoconjugate in lymphoma-bearing nude mice.

FIG. 3 illustrates the effects of Lym-1/IL-2 vasoconjugate predoseescalation on tumor uptake by I-125 Lym-1 F(ab′)₂ tracer.

FIG. 4 illustrates the effects of time of administration of Lym-1/IL-2vasoconjugate in lymphoma-bearing nude mice.

SUMMARY OF THE INVENTION

The invention provides immunoconjugates comprising biologically activeagents, capable of provoking a vasoactive response, which are linked tomonoclonal antibodies (mAbs). The mAbs have the ability, whenadministered in vivo to a host, of preferentially binding to neoplastictissue such as tumor cells or tumor cell ghosts. The biologically activeagent is in this way localized at the site of neoplastic tissue where itprovokes a response in which, by means of vasodilation and increasedvasopermeability, or through the mechanism of an inflammatory response,the local circulation and blood supply to the tumor tissue are improved.The expansion of the blood volume within the tumor allows therapeuticand diagnostic agents, subsequently introduced into the host, topenetrate the tumor more completely and thus to be delivered in a largerand more effective dose.

The use of effective vasoactive conjugates preliminary to some types ofimmunotherapy will not only potentiate that therapy, but substantiallyreduce the risk of deleterious side effects in the use of antibodyconjugates comprising cytopathic substances such as anti-neoplasticdrugs, toxins, or radionuclides. Such antibody conjugates remainingunbound in the circulation may lead to the unintended destruction ofnormal tissue, particularly tissues of organs of the renal, hepatic andreticuloendothelial systems which must eliminate them from the body. Byincreasing the relative amount of dose which can bind to the targettumor, vasoactive conjugates make it possible to use a lower effectivedose thereby reducing the amount of unbound circulating cytotoxic agent,and the risk to normal tissue.

In addition to the immunoconjugates discussed above, which were alsodisclosed in our above-referenced, prior application, this inventionprovides conjugates comprising vasoactive agents linked to monoclonalantibodies or, in a further extension, to other moieties (e.g.,macromolecules or liposomes) that localize to permeable vessels.Therefore, while the term “immunoconjugates” may be used throughout thedisclosure, it is to be expressly understood that a conjugate comprisedof at least one immunoactive moiety is but one example of the diversearray of conjugates contemplated by the present invention.

The monoclonal antibodies are selected for the ability, whenadministered intravenously (“I.V.”), of preferentially binding to tumorsor to blood vessels and related structures in areas of inflammation, orwhere blood vessels are structurally abnormal at the tumor site. Thevasoactive agent is in this way localized selectively at the sites oftumor or inflammation, where it provokes further increases inpermeability. Such increases are selective for the sites and serve tofacilitate passage of subsequently administered I.V. therapeutic agentsfrom blood to tissue at the sites.

Selective permeability enhancement, induced by these vasoactive antibodyconjugates, serves to increase the portion or dose of agentsadministered I.V. that reach the site of desired therapeutic action.This will not only potentiate therapy, but will substantially reduce therisk of deleterious side effects due to toxic metabolites or due to thedevelopment of immunological hypersensitivity responses.

According to one aspect of the invention, there are providedpharmaceutical conjugate, comprising a delivery vehicle having theability to localize at the site of neoplastic tissue, and an agent boundto the delivery vehicle acting to increase the blood supply to theneoplastic tissue. In a preferred aspect, the conjugate is of sufficientsize to be unable to penetrate normal, healthy vascular endothelium, butable to penetrate the vascular endothelium of tumor tissue. In anothervariation, the agent acts to increase vasopermeability at an active sitein vascular endothelium, or acts to provoke or exacerbate a localinflammatory reaction at an active site in vascular endothelium. Anothervariation of the present invention suggests a vasoconjugate incombination with an antineoplastic radioisotope or an antineoplastictoxin. In another embodiment, the delivery vehicle comprises amacromolecule or particle having a molecular weight (MW) between 30,000and 200,000.

In another aspect, the present invention suggests a method for thediagnosis of neoplastic tissue, comprising administering to a hosthaving the tissue an effective amount of a delivery vehicle having theability to concentrate at the site of the tissue, the antibody beingconjugated to an agent which acts to increase the blood supply to theneoplastic tissue, and contemporaneously or thereafter administering tothe host a tumor imaging agent. In another embodiment, the diagnosticagent is administered as a conjugate, comprising a delivery vehiclehaving the ability to concentrate at the site of the tissue, conjugatedto the tumor imaging agent.

According to another aspect of the invention, there are providedimmunoconjugates comprising monoclonal antibody having the ability tolocalize to tumors or in the vicinity of structurally abnormal bloodvessels, new vessels, or inflamed blood vessels at the tumor site; theseconjugates further contain selected vasoactivators. In one preferredembodiment, the monoclonal antibody has specificity for subendothelialcomponents of the blood vessel wall that become accessible tocirculating antibody in inflamed vessels and in structurally abnormalvessels such as those found in tumors. Such target antigens includefibronectin, laminin, and type IV collagen. In another embodiment, theantibodies have specificity for components of the coagulation cascadethat are activated in the wall, or in the immediate perijacentenvironment of inflamed blood vessels, or in the necrotic areas of thetumor. Such antigens include fibrin, thrombin, and components of thecomplement system, and antibodies are available with thesespecificities. Yet another embodiment would employ antibody withspecificity for antigens selectively expressed upon endothelial cells ininflamed blood vessels, but not in normal vessels. Such antigens wouldinclude various cell adhesion molecules that have been identified asresponsible for adherence of polymorphonuclear leukocytes to inflamedblood vessel walls. The blood coagulation product fibrin is aparticularly favored target for this approach. Fibronectin, which isdistributed in a subendothelial distribution in blood vessels and isrevealed by structural abnormality or by permeability change, is anotherfocused target for this approach.

Another embodiment suggested by the present invention is the chemicallinkage of a vasoactive moiety to a mAb with specificity to the tumor.In this instance, the mAb would act to locate the vasoactive moiety tothe tumor site during or after binding to tumor cells within the tumor.The vasoactive moiety would then act on the surrounding vessels in theimmediate area of mAb binding.

Another embodiment suggested by the present invention is the linkage ofa delivery vehicle to a vasoactive agent at the molecular level—i.e.,via the construction of a “cassette” to be inserted into an organism,said cassette including, at a minimum, the genes coding for the deliveryvehicle and the vasoactive peptide. The cassette could, in anotherembodiment, also include regulatory sequences. The cassette could beinserted into the genome of the organism, into a plasmid, or into avector such as a virus or retrovirus, for example.

This invention further discloses at least three antigen markers fortumor vasculature or “leaky” vasculature that emulates new tumorvasculature, and suggests means of utilizing same to construct deliveryvehicles capable of specific localization to tumor sites, inflamedtissues, abscesses, and similar sites containing “leaky” vessels.

Therefore, according to one aspect of the invention, there are providedimmunoconjugates comprising a monoclonal antibody having the ability tolocalize at the site of neoplastic tissue (mAb), and a vasoactive agentbound thereto. In a preferred embodiment, the mAb has a specificity fortumor cells, and in a particularly preferred embodiment, the mAb hasspecificity for antigens associated with B-cell lymphoma cells.According to this embodiment, the monoclonal antibody may be Lym-1 orLym-2.

According to one embodiment, the vasoactive agent comprises a peptide,and in a preferred embodiment the peptide is a tachykinin. In aparticularly preferred embodiment, the tachykinin is selected from thegroup consisting of phyllomedusin, physalaemin, and substance P.

According to another preferred embodiment, the vasoactive peptidecomprises a leukotriene. In a particularly preferred embodiment, theleukotriene is selected from the group consisting of B4, C4, D4, and E4.

According to another preferred embodiment, the vasoactive peptidecomprises an anaphylatoxin. In a particularly preferred embodiment, theanaphylatoxin is selected from the group consisting of C3a and C5a.

According to yet another embodiment, the vasoactive peptide is alymphokine. In a particularly preferred embodiment, the lymphokine isselected from the group consisting of interleukin-1, interleukin-2 andtumor necrosis factor.

In another preferred embodiment, the vasoactive peptide is thechemotactic factor ECF-A.

In yet another preferred embodiment, the vasoactive peptide is aninflammagen. In particularly preferred embodiments, the inflammagen isselected from the group consisting of mastoparan and bestatin.

In yet another preferred embodiment, the vasoactive peptide is aprotease. In particularly preferred embodiments, the protease isselected from the group consisting of trypsin, chymase and thrombin.

In yet another preferred embodiment, the vasoactive agent is avasoactive carbohydrate. In particularly preferred embodiments, thecarbohydrate is selected from the group consisting of glucan andproteoglucans.

In yet another preferred embodiment, the vasoactive agent is a lipid. Inparticularly preferred embodiments, the lipid is selected from the groupconsisting of platelet-activating factor and prostaglandins.Alternatively, the lipid may be derivatized as the drug, Viprostol.

In yet another embodiment of the invention, the vasoactive agent is abiological amine. In a particularly preferred embodiment, the amine ishistamine.

According to another aspect of the invention, the mAb of theimmunoconjugate may be an intact immunoglobulin. In a preferredembodiment, the mAb may be an immunoglobulin fragment consisting of themonovalent HL isoform. In another preferred embodiment, the mAb is onefrom which the Fc portion has been removed. In a particularly preferredembodiment, the mAb is in the form of the F(ab′)2 portion.

According to another aspect of the invention there is provided a methodfor treating a tumor, comprising administering a vasoactiveimmunoconjugate to a tumor host, wherein the immunoconjugate comprises amAb or other delivery vehicle having the ability to localize at the siteof neoplastic tissue, allowing the immunoconjugate to bind to tumortissue and allowing for the vasoactive effect of the immunoconjugate tooccur, and either simultaneously or thereafter administering atherapeutic agent to the tumor host. In a preferred embodiment, theadministered therapeutic agent is a cytotoxic chemical agent. In aparticularly preferred embodiment, the administered therapeutic agent isa cytotoxic immunological agent.

In another embodiment there is provided a method for diagnosing a tumor,comprising administering a vasoactive immunoconjugate to a tumor host,wherein the immunoconjugate comprises an mAb having the ability toconcentrate at the site of neoplastic tissue, allowing theimmunoconjugate to bind to tumor tissue and allowing for the vasoactiveeffect of the immunoconjugate to occur, and then eithercontemporaneously or thereafter administering to the host animmunodiagnostic agent.

Moreover, according to another aspect of the present invention, thereare provided conjugates comprising a delivery vehicle having the abilityto localize at the site of neoplastic tissue, and an agent bound to thedelivery vehicle, the agent acting to potentiate the action of adifferent antineoplastic agent against the tissue by increasing theblood supply thereto. Another embodiment suggests a conjugate ofsufficient size to be unable to penetrate normal, healthy vascularendothelium, but able to penetrate the vascular endothelium of tumortissue.

In another embodiment, the agent acts to increase vasopermeability at anactive site in vascular endothelium, while yet another embodiment of thepresent invention suggests that the agent acts to provoke or exacerbatea local inflammatory reaction at an active site in vascular endothelium.

In various embodiments of the disclosed invention, the agent maycomprise, for example, a drug, a vasoactive peptide, a biological amine,or a pharmaceutical compound. Similarly, the conjugate may comprise, forexample, a carbohydrate, such as a glucan or proteoglucan, or it maycomprise a lipid, such as platelet activating factor or prostaglandins.

Another aspect of the present invention provides a delivery vehicle witha specificity for molecules that are selectively expressed in vascularendothelium that is damaged, inflamed or structurally abnormal.Preferred delivery vehicles include, without limitation, the F(ab′)₂,F(ab), or HL fragments of an immunoglobulin molecule, dextrans,monoclonal antibody, or liposomes. In especially preferred embodiments,the liposomes have a diameter on the order of 80 nm, and the dextransare high molecular weight dextrans (70-150 KD). In an even morepreferred embodiment, the dextrans selectively localize in the walls ofpermeable vessels.

In another embodiment, the delivery vehicle has specificity forsubendothelial components of the blood vessel wall that becomeaccessible to circulating antibody in inflamed vessels and instructurally abnormal vessels such as those found in tumors. Suggestedcomponents include, without limitation, fibronectin, laminin, and typeIV collagen.

A further embodiment discloses a delivery vehicle with specificity forcomponents of the coagulation cascade that are activated in vascularwalls, in the immediate perijacent environment of inflamed bloodvessels, or in the necrotic areas of the tumor. In another preferredembodiment, the components comprise fibrin, thrombin, and components ofthe complement system.

In yet another embodiment, the delivery vehicle has specificity forantigens selectively expressed in or upon endothelial cells in inflamedvascular tissue such as that found in the vicinity of tumors, but not innon-inflamed vascular tissue. Antigens suggested by the presentinvention include cell adhesion molecules responsible for adherence ofpolymorphonuclear leukocytes to inflamed vascular tissue, fibrin,fibronectin, fibrin degradation products, cell enzymes, platelets, andplatelet products. In a further embodiment, the enzymes includeperoxidases or other proteins that are released in necrotic or inflamedtissues.

The present invention also suggests a method for the treatment ordiagnosis of neoplastic tissue, comprising administering to the host ofthe tissue an effective amount of a delivery vehicle having the abilityto concentrate at the site of the tissue, the antibody being conjugatedto an agent which acts to potentiate the action of a differentantineoplastic agent against the tissue by increasing the blood supplythereto, and contemporaneously or thereafter administering to the host asecond conjugate, comprising a delivery vehicle having the ability toconcentrate at the site of the tissue, conjugated to a therapeutic ordiagnostic agent.

Another embodiment suggests a method for immunotherapy of neoplastictissue, comprising administering to a tumor host an effective amount ofa conjugate referred to herein, and contemporaneously or thereafteradministering to the tumor host a delivery vehicle having the ability toconcentrate at the site of the tissue and directed to the therapythereof. A further embodiment discloses the conjugation of a tumoricidalagent to the delivery vehicle.

Still another embodiment suggests a method for the immunotherapy ofneoplastic tissue, comprising administering to a host of the tissue aneffective amount of a conjugate referred to herein, andcontemporaneously or thereafter administering to the host apharmacological agent directed to the therapy of the tissue.

In a further embodiment, a method for the immunodiagnosis of neoplastictissue, is disclosed, said method comprising administering to a host ofthe tissue an effective amount of a conjugate referred to herein, andcontemporaneously or thereafter administering to the host a secondconjugate comprising a delivery vehicle having the ability toconcentrate at the site of the tissue and a detectable agent conjugatedthereto.

Another embodiment discloses a method for immunotherapy of inflamedtissue, comprising administering to a tumor host an effective amount ofa conjugate referred to herein, and contemporaneously or thereafteradministering to the tumor host a second delivery vehicle having theability to concentrate at the site of the tissue and directed to thetherapy thereof.

A method for the immunotherapy of inflamed tissue is also disclosedherein, comprising administering to a host of the tissue an effectiveamount of a conjugate referred to herein, and contemporaneously orthereafter administering to the host a pharmacological agent directed tothe therapy of the tissue.

Another aspect of the present invention suggests a method forconstructing a conjugate for pharmaceutical use, comprising attaching adelivery vehicle having the ability to localize at the site ofneoplastic tissue or nucleotides coding for same, to at least one agentacting to increase the blood supply to the neoplastic tissue ornucleotides coding for same. The present invention further suggests atherapeutic kit, comprising a conjugate, comprising a delivery vehiclehaving the ability to localize at the site of neoplastic tissue, and anagent bound to the delivery vehicle acting to increase the blood supplyto the neoplastic tissue, and an antineoplastic therapeutic agent.Additionally, the present invention discloses a diagnostic kit,comprising a conjugate, comprising a delivery vehicle having the abilityto localize at the site of neoplastic tissue, and an agent bound to thedelivery vehicle acting to increase the blood supply to the neoplastictissue, and a tumor imaging agent.

Finally, another embodiment of the present embodiment suggests a methodfor genetically constructing a conjugate, comprising attaching at leastone agent or nucleotides coding for same to at least one deliveryvehicle or nucleotides coding for same.

These and other advantages and features of the present invention willbecome more fully apparent from the following description and appendedclaims.

DETAILED DESCRIPTION

Systemically administered vasoactive agents have been shown to inducemore extensive changes in tumor vessels than in normal vessels. (See,e.g., Cater, et al., Br. Cancer 20: 517 (1966).) This effect can bemaximized by linking vasoactive agents to monoclonal antibodies or othermoieties that bind with molecules in the vascular wall, or in theimmediate surrounding environment, of abnormal blood vessels withintumors. This application is thus an extension of our previousapplication, as cited above, in which antibody with specificity fortumor cells was conjugated with vasoactive agents, with the goal ofinducing permeability changes. The present application differs inrecognizing that permeability changes are more effectively achieved byutilizing antibodies with specificity for components of the blood vesselwall, or other molecules in the immediate perivascular environment, asan alternative to the use of antibody against tumor cells, which may besome distance removed from the blood vessels and, therefore, are not“seen” by antibodies circulating in the bloodstream.

Preferably, the antibodies to be used have the following properties.First, following chemical conjugation with various vasoactive agents,they retain the ability to bind with antigen. Second, they do not bindwith any component of blood or normal, intact, non-inflamed endothelium.Third, they show little or no tendency to pass across the endothelium ofnormal blood vessels from blood into tissues. Fourth, they bind tomolecules that are selectively expressed in, or adjacent to, bloodvessels that are inflamed or structurally abnormal, as are many vesselsin tumors. Finally, upon binding, the conjugated antibody deliversvasoactive compounds directly to the active site in the blood vesselwall. The explosive permeability changes that follow favor furtherbinding of monoclonal antibody at the site, thereby establishingphysiologic changes in the tumor vessels, while normal vessels areunaffected.

Immediately following induction of this localized permeability changeand/or increase in tumor blood flow, a potential therapeutic agent, suchas a drug or a monoclonal antibody, injected intravenously, showspreferential passage from the blood into tissue fluid at the abnormallypermeable site. By this mechanism, the percentage of a given dose ofagent delivered to the tumor site has been multiplied from two to sixfold in studies to be described within. This method may be utilized forimproving delivery of anticancer agents to tumor sites, either drugs,monoclonal antibodies, or conjugates of monoclonal antibodies withdrugs, toxin or radioisotopes.

Alternatively, other moieties such as high molecular weight dextrans(i.e., 70-150 kilodaltons, KD) that selectively localize in the walls ofpermeable vessels may be used in lieu of monoclonal antibodies asdelivery vehicles for the vasoactive agents. (See, e.g., Dvorak, et al.,Am. J. Pathol. 133: 95-109 (1988).) In further examples, liposomes witha diameter on the order of 80 nanometers (nm) are disclosed as showingpreferential passage across permeable vessel walls in tumors and mayalso be used as delivery vehicles for permeability-enhanced therapy.(For a discussion of the use of liposomes as drug carriers in cancertherapy, see Weinstein, J. N., Cancer Treatment Rep 68: 127-134 (1984).)

The same considerations apply to: 1) the delivery of antibody-isotopeconjugate to tumor sites for the purpose of obtaining improvedradioimaging; 2) the delivery of antimicrobial agents to sites ofinflammation caused by infectious agents, in order to increase theconcentration of agent in the vicinity of the organism withoutincreasing the overall dose to the patient; and 3) the delivery ofvarious anti-inflammatory drugs to the site of acute or chronicinflammation throughout the body, for the purpose of suppressing theadverse affects of inflammation. In each instance, I.V. administrationof the designated therapeutic agent is preceded by an I.V. injection ofthe antibody-vasoactive agent conjugate, designed to produce transientpermeability enhancement of the desired site of action of thetherapeutic agent.

A further embodiment employs monoclonal antibodies to macromoleculesthat are exposed to the bloodstream in structurally abnormal vesselswithin necrotic areas in tumors or inflamed tissues. Such antigensinclude fibrin degradation products, and various cell enzymes such asperoxidases that are released by granulocytes or other cells in necroticor inflamed tissues.

The various vasoactive compounds for attachment to antibody areanalogous to those described below, and include peptides, carbohydrates,lipids, and their derivatives.

Another embodiment would employ antibody with specificity for antigensselectively expressed upon endothelial cells in inflamed blood vessels,but not in normal vessels. Such antigens would include various celladhesion molecules that have been identified as responsible foradherence of polymorphonuclear leukocytes to inflamed blood vesselwalls. The blood coagulation product fibrin is a particularly favoredtarget for this approach. Fibrin is not normally present within thebloodstream, existing only as a circulating precursor molecule,fibrinogen, which has a molecular weight (MW) of approximately 340kilodaltons (KD). Likewise, fibrin is not present in normal tissue ortissue fluids. Fibrinogen is also absent from tissue fluids, since itshigh molecular weight precludes escape from the blood across normal,intact endothelium.

In the presence of endothelial damage or increased permeability,fibrinogen may, however, escape into the tissues where it is rapidlyconverted to fibrin through activation of intravascular clottingmechanisms. Fibrin deposits thus form at the site of permeabilitychange. In tumors, microdeposits of fibrin are particularly present incapillary sprouts and in the vicinity of blood channels that lackcomplete endothelial lining.

Furthermore, fibrinogen serves as a marker of vascular leakage by virtueof its molecular weight characteristics. Secondly, its detection isfacilitated by its conversion into an insoluble product immediately uponescape from the vessel. Monoclonal antibodies directed against fibrin(that are non-reactive with fibrinogen) will therefore show selectivehoming to permeable vessels that have been “marked” by fibrinogenleakage and fibrin deposition.

Fibronectin, which is distributed in a subendothelial distribution inblood vessels and is revealed by structural abnormality or bypermeability change, is another focused target for this approach. See,e.g., Christensen, et al., Cancer (1988); Dvorak, et al., NEJM 315: 1650(1986); and Jain, Cancer Res. 48: 2641 (1988).)

Other embodiments of vasoactive conjugates may also prove efficacious,including those which improve the extravascular penetration and bindingof monoclonal antibodies, as well as other drugs or molecules. Just asthe conjugates disclosed herein have proven effective when largemolecules are utilized, smaller molecules, such as chemotherapeuticdrugs, may also exhibit increased penetration and binding.

Embodiments using vehicles other than monoclonal antibodies employmacromolecules (molecular weight range: 70,000-1,000,000 or more) ormicroparticles, including liposomes, with a diameter on the order of 80nanometers (nm) that localize to permeable vessels on the basis of theirphysio-chemical characteristics. In one example, dextrans (MW 150 KD)are conjugated with vasoactive agents and serve to deliver biologicallyactive molecules to vessels that show marginal permeability changes,thereby markedly enhancing permeability at the sites only. As a result,therapeutic modalities administered subsequently show a higherproportion of administered dose at the initial sites.

The immunoconjugates of the invention are prepared by geneticapproaches, or covalently or otherwise linking a selected clinicallyuseful mAb to a selected biologically active agent which is inflammationprovoking, and preferably vasoactive. The linking agent and the chemicalprocedure of assembling the immunoconjugate should be selected andcarried out so as not to compromise the effectiveness of the antibody inbinding to target cells or the effectiveness of the vasoactive agent instimulating natural defense mechanisms.

Selection of Delivery Vehicles

1. Monoclonal Antibodies

Suitable monoclonal antibodies for use in the invention comprise notonly those having a specificity for antigens unique to the tumor cells,but also those having a shared specificity for antigens of normaltissues. The essential property is that these monoclonal antibodies beeffective, according to the purpose of the invention, as carriers whichpreferentially concentrate vasoactive agents at the site of the tumor.Suitable monoclonal antibodies may be those having a specificity toantigens, such as intercellular substances, that are either moreabundant or more easily bound in tumor tissue than in normal tissue. Oneexample is antibody to nuclear antigens, as disclosed in U.S. Pat. No.4,861,581.

Some mAbs against tumor or normal cellular antigens, suitable for use inthe immunoconjugates of the invention, are available commercially(Centocor, Malvern, Pa.; Hybritech, San Diego, Calif.). Others may beprepared according to the well-established hybridoma procedure of Kohlerand Milstein, (Nature 256: 495 (1975)), and commercial kits facilitatethis process. To prepare hybridoma cell lines, splenocytes from miceimmunized with tumor antigen are fused with cells from a non-secretingmouse mycloma fusion line, such as P3X63-Ag8.653 (American Type CultureCollector, Rockwell, Md.), according to kit instructions, for example,HyBRL Prep Kit (Bethesda Research Labs, Bethesda, Md.). The fusedhybridomas cells are then transferred into the wells of microtiterplates where they are grown for several days. The supernatants in thewells are tested for production of mAbs to tumor or cellular antigens byany convenient immunoassay, for example, an ELISA, and the positivehybridoma cell lines, that is, those producing acceptable mAbs, areexpanded into permanent culture. MAbs may be purified from thesupernatants of these cultures by gel chromatography, for example, usingthe Affi-Gel Protein A column (Bio-Rad, Richmond, Calif.).

In a preferred embodiment of the invention, commercially available mAbsspecific for lymphoma cells, Lym-1 and Lym-2, are used (TechnicloneInternational, Inc., Tustin, Calif.).

The suitability of tumor-specific mAbs for in vivo use is determined bythe biodistribution, cellular localization, selective binding, and rateof clearance from the tumor host, or an animal model of the tumor host.The performance of the assembled immunoconjugates may also be determinedby parallel studies. Studies to assess this suitability are convenientlycarried out by means of labeled mAbs, for example, ¹³¹I-mAbs,radioiodinated, for example, by the modified Chloramine-T procedure ofMcFarlane, A., Biochem. J. 62: 135-143 (1956).

The immunoreactivity of radiolabeled anti-tumor mAbs may be determinedby an in vitro live cell radioimmunoassay procedure as described inExample 1 for the Lym-1 and Lym-2 mAbs (see Epstein, A. et al.,“Malignant Lymphomas and Hodgkin's Disease: Experimental and TherapeuticAdvances,” Martinus Nijoff Publ. Co., Boston (1985), pp. 569-577).

The effectiveness of an anti-tumor mAb in vivo may be evaluated byappropriate radioimaging, biodistribution, histological studies, andautoradiographic methods performed after injecting the tumor-bearinghost with the labeled mAb.

The ability of the mAb to concentrate selectively at the tumor site isdetermined by radioimaging. Posterior gamma scintillation images(100,000 cpm) are obtained from the anesthetized host on alternate daysafter injection of the radiolabeled mAb, using a gamma scintillationcamera with a pinhole collimator. The camera is preferably interfaced toa computer system. An appropriate, ¹³¹131 I standard with the sameactivity is counted to quantitate the data.

At an optimal time, as indicated by the imaging studies, the host animalis sacrificed and blood, major organs and tumor tissue excised, weighed,and counted to determine the biodistribution of the mAb. Further, tumortissue may be fixed and embedded, and tissue sections examined byautoradiography to determine the radiolabeled mAb bound to the tumor.

The mAb of the immunoconjugate may be either intact whole antibody, themonovalent HL isoform, the F(ab′)₂ portion of antibody, or Fab antibodyfragments. Removal of all or part of the Fc portion of the antibodymolecule can facilitate its use by removing sites or domains whichinteract with non-tumor components such as Fc receptors or complementwhile leaving the antigen binding sites intact. Antibody fragments suchas Fab, HL, and F(ab′)₂, which have ⅓, ½ and ⅔ the weight of wholeantibody respectively, have the ability to cross capillary walls anddiffuse through the interstitial tissue more readily, and so are able todiffuse more rapidly into the tumor. On the other hand, however, theFab, HL, and F(ab′)₂ fragments are cleared from the circulation morerapidly. Wilbonk et al., Cancer 48: 1768-1775 (1981) found higher tumorto organ binding ratios with Fab fragments, but a 3-fold higher absoluteconcentration in the tumor with whole antibody. Wahl et al., J. Nucl.Med. 24: 316-325 (1983), in studies using monoclonalanti-carcinoembryonic antigen (CEA), found that F(ab′)₂ fragments werethe best compromise between the rapidly cleared Fab fragments and theslowly cleared whole antibody. Fab fragments may be prepared bydigestion of whole antibody with papain, or digestion of whole antibodyto F(ab′)₂ fragments with pepsin, followed by digestion of interchaindisulfide bonds to yield univalent fragments. (See Porter, R., Biochem.J. 73: 119 (1959).) HL fragments may be derived according to thetechnique set forth in Nature 194: 355 (1962) or PNAS (USA) 50: 314-321(1963).

2. Macromolecules or Microparticles

Liposomes and macromolecules such as dextran are selected on the basisof their ability to localize to tumors, as detected by radioimaging inexperimental models. The methods used are analogous to those describedabove for monoclonal antibodies.

Selection of Vasoactive Agents

The vasoactive immunoconjugates of the present invention are distinctfrom drug or toxin immunoconjugates in their mode of action. Drug andtoxin conjugates are used to kill tumor cells directly. Vasoactiveconjugates are used to increase the flow of blood and/or the vesselpermeability in the tumor so as to improve the extravascular penetrationand binding of monoclonal antibodies and other drugs or molecules invivo. They may act directly by increasing the volume of tumor blood flowor the degree of tumor blood vessel “leakiness,” or indirectly byinducing an inflammatory immune response at the tumor site. Inflammationcan be induced by chemotactic factors which attract polymorphonuclearleukocytes, macrophages, eosinophils, basophils, mast cells, T-cells andother cells associated with inflammation. These cells, when stimulated,secrete immunomodulatory factors which then act on the tumor blood flowand blood vessel permeability to increase the percent of the injecteddose penetrating and binding to the tumor.

Vasoactive agents having the described reactivity at the tumor site andsuitable for linking to monoclonal antibodies in an immunoconjugate arefound in several biochemical classes, including peptides, carbohydrates,and lipids, and their derivatives.

Peptides, either natural, synthetic, or recombinant, comprise the mostabundant source of vasoactive agents suitable for use inimmunoconjugates.

Tachykinins are a family of deca-, enceda-, and dodeca-peptide amides,having a phenylalanine (Phe) residue at position 5 from the COOHterminus. They have potent pharmacological effects on blood pressure,non-vascular smooth muscles, and the exocrine glands (Erspamer, V.,TINS, November 1981, pp. 267-269). Substance-P, a mammalian tachykinin,promotes vasodilation and plasma extravasation through antidromicstimulation of chemosensitive nerve fibers (Lambeck, F. and Halzer, P.,Naunyn-Schmeideberg's Arch. Pharmacol. 310: 175-183 (1979)). Substance-Palso mediates histamine release from tissue mast cells (Hagermark, O. etal., J. Invert. Dermatol. 71: 233-235 (1978)). In preferred embodimentsof the invention, Substance-P and an amphibian analog, physalaemin, areconjugated to clinically useful Mabs for use in promoting the dilationof the tumor microvasculature.

The leukotrienes are sulfidopeptides which are potent mediators inatopic allergy. The action of these mediators on blood vessels with itsassociated inflammatory action is responsible for the clinicalmanifestations and physical features of the disease. As little as 1 nmolof leukotrienes C₄, D₄ or E₄ elicits erythema and wheal formation. Inpreferred embodiments of the invention, leukotrienes B₄, C₄, D₄ and E₄are conjugated to clinically useful mAbs for use in producing a localinflammatory reaction at the tumor site.

Anaphylatoxins are peptide fragments released during activation of serumcomplement. Enzymatic cleavage of complement proteins C3 and C5 releasesactivation peptides C3a and C5a, respectively. These peptides have beendesignated anaphylatoxins because of their ability to produce a reactionthat resemble anaphylactic shock. Both C3a and C5a have the ability toincrease vascular permeability and to release granules containingserotonin and histamine from tissue mast cells. C5a, in addition andperhaps cooperatively with C3a, is chemotactic, inducing the migrationand aggregation of neutrophils. (See Nagata, S. et al., Int. Arch.Allergy Appl. Immun. 82: 4-9 (1987).) In preferred embodiments of theinvention, C3a, C5a, or their biologically active peptide sequences,either singly or in combination, are conjugated to tumor-specific Mabsand used to produce a localized inflammatory response at the tumor siteas an alternative approach to enhance the extravascular penetration ofmonoclonal antibodies.

The biological activity of these peptides can be reproduced by syntheticoligopeptides, 8 to 21 amino acids in length, which contain residuescommon to native C3 at its COOH terminus end. (See, e.g., Hugli, T. andErickson, B., PNAS USA 74: 1826-1830 (1977).)

Lymphokines, comprising the interleukins IL-1 and IL-2 and tumornecrosis factor (TNF), are endogenous stimulators of the immune responsethat act and interact in complex roles in the defense of an organismagainst foreign agents. (See, e.g., Kampschmidt, R., J. Leukocyte Biol.36: 341-355 (1984).)

IL-2 is of particular interest for use in immunoconjugates. Thislymphokine has no anti-tumor activity of its own, but appears to havepotent activity when administered with lymphokine-activated killer (LAK)cells. Its use as an anti-tumor agent appears to be limited, because itsability to mediate vascular permeability and extravasation in the hostproduces severe side effects due to retention of fluid. (See Fairman, R.et al., Cancer Res. 47: 3528-3532 (1987); Rosenstein et al., J. Immunol.137: 1735-1742 (1986).) However, the vasoactive properties of IL-2 arewell suited to its use in the immunoconjugates of this invention. SinceIL-1 stimulates production of IL-2 from lymphocytes, and TNF appears toexert synergistic properties in conjunction with other lymphokines,their immunoconjugates could be useful in combination with those ofIL-2. (See Talmadge et al., Cancer Res. 47: 2563-2570 (1987); Philip, R.and Epstein, L., Nature 323, September 4, pp. 86-89 (1986).) As in thecase of the C3a anaphylatoxin, small synthetic oligopeptides, comprisingthe functional region of interleukin, can also be suitable for use inthe immunoconjugates. (See, e.g., Antoni, G. et al., J. Immunol. 137:3201-3204 (1986).)

Yet another group of peptides suitable for use in the vasoactiveimmunoconjugates are the human eosinophil acidic tetrapeptides (ECF-A),Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu, which have the ability throughchemotaxis to promote a local cosinophilia (Turnball, L. et al.,Immunology 32: 57-63 (1977)).

Further, certain peptides, the inflammagens, when used in vasoactiveimmunoconjugates, would be capable of degranulating mast cells at thetumor site, releasing histamine and provoking a local inflammatoryresponse. One such inflammagen, mastoparan, is a tetradecapeptideisolated from wasp venom (Okano, Y. et al., Fed. Europ. Biochem. Soc.188(2): 363-366 (1985)). In a preferred embodiment, mastoparan, eitherisolated from the natural source or produced synthetically is linked toa tumor-specific mAb. (See Hirai, Y. et al., Chem. Pharm. Bull. 27(8):1942-1944 (1979).)

Proteases released from mast cells upon immunologic activation appear toprovoke hypersensitivity reactions in skin. The possible actions ofthese proteases include digestion of the blood vessel basement membranewith resultant increased vascular permeability and the influx ofsecondary inflammatory cells. Tryptase, an endopeptidase similar topancreatic trypsin, is a tetramer composed of two 35 kilodalton and two37 kilodalton subunits. It is the principal protease of human lung mastcells and is present in mast cells from all locations. Chymase, found inhuman skin mast cells, has a specificity like that of pancreaticchymotrypsin. (Serafin, W. and Austin, K., NEJM, July 2, pp. 30-34(1987).) In preferred embodiments of the invention, tryptase and chymaseare conjugated to tumor-specific mAbs for use in producing a localinflammatory reaction at the tumor site.

Certain lipid compounds can be effective as immunoconjugates. In oneembodiment of the invention, platelet-activating factor (PAF) is thevasoactive agent of the immunoconjugate. PAF is a phospholipid producedby human neutrophils which appears to be a potent mediator of the immuneresponse. (See Braquet, P. and Rola-Pleszezynski, M., Immunology Today8(11): 345-352 (1987).) PAF is linked with virtually all inflammatoryand immune processes, for example, with respect to the vasoactivepeptides listed above, PAF stimulates Substance-P release, and inducesthe formation of other vasoactive agents, such as leukotrienes orprostaglandins. Its use in immunoconjugates can amplify the effect ofthese other agents whether endogenous or used in complementaryimmunoconjugates.

In yet another embodiment of the invention, the hypotensive agent,Viprostol, a prostaglandin derivative, (American Cyanamid, Pearl River,N.Y.) is the active agent in the immunoconjugate. Viprostol lowersarterial blood pressure mainly through vasodilation. (See Chan, P. etal., J. Hypertension 4(6): 741-746 (1986).) Use of a tumor-specific,targeted Viprostol dose will dilate the vasculature of the tumor toexpand blood volume therein.

Similarly, in other embodiments, the natural prostaglandins, (PGE's), orsynthetic analogues which are known to possess hypotensive effects, canbe effectively used. (See Birnbaum et al., J. Medicinal Chem. 25(5):492-494 (1982).)

Histamine, a component of mast cell granules released upon immunestimulation, acts through two types of receptors, designated H₁, and H₂,to produce, among other effects, increased venular permeability andvasodilation as described for the leukotrienes. (Serafin, W. and Austin,K., NEJM, July 2, pp. 30-34 (1987).) In preferred embodiments of theinvention, histamine is conjugated to tumor-specific mAbs for use inproducing a local inflammatory reaction at the tumor site.

In yet other embodiments of the invention, the effective agents of theimmunoconjugates are vasoactive carbohydrate compounds. In a preferredembodiment, the vasoactive carbohydrate is glucan. Glucan is a.beta.-1,6 linked polyglucose derived from Saccharomyces cerevisiaewhich has a number of immunopotentiating effects (Glovsky, M. et al., J.Reticuloendothelial Society 33: 401-413 (1983)), but, unlike theinterleukin IL-2, is non-toxic. (See Sherwood, E. et al., J. BiologicalResponse Modifiers 7: 185-198 (1988).) Glucan appears to exert itseffects by stimulating the complement system, generating, among othercomplement fragments, the vasoactive C3a and C5a peptides. Glucan,targeted to tumors by means of specific mAbs, could act locally throughC3a and C5a to dilate the tumor vasculature.

Conjugant molecules are selected according to availability andapplicability to the stated goals of therapy or study.

Chemical Conjugation Methods

The structural link between the mab, macromolecule, or microparticle andthe vasoactive agent, and the chemical method by which they are joined,should be chosen so that the binding ability of the mAb and thebiological activity of the agent, when joined in the conjugate, areminimally compromised.

Among the methods from which the most effective conjugation chemistrymay be selected are the following:

a) Carbodiimides may be regarded as anhydrides of ureas.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (ECDI) producescrosslinks between the antibody and the conjugant, regardless of eithermolecule's orientation. Conjugants are derived by condensation of theantibody and conjugant under acidic conditions with ECDI. This methodprovides a rapid and simple means of conjugation. (See Goodfriend, T. etal., Science 144: 1344-1346 (1964).) The use of ECDI to join physalaeminor Interleukin-2 to Lym-1 or Lym-2 is illustrated in Examples 2 and 7.

b) N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) is aheterobifunctional reagent which introduces thiol groups to the terminalamino of proteins, and has been used in a number of immunoconjugates.(Carlsson, J. et al., Biochem. J. 173: 723-737 (1978).)

c) The use of SMCC method to conjugate C3a to F(ab′)₂ fragments of mabsis illustrated in Example 3.

d) The cis-aconitic linkage described by Shen, et al has thecharacteristic of releasing conjugant at low pH, such as in a secondarylysosome following endocytosis of the receptor-bound antibody molecule.The method allows conjugation to the carbohydrate side groups of theantibody molecules. (Shen, W.-C., and Ryser, H., Biochem. Biophys. Res.Comm. 102(3): 1048-1054 (1981).) The use of cis-aconityl derivatizationto conjugate the drug Viprostal to an mAb is illustrated in Example 4.

e) Periodate oxidation can be used to oxidize and cleave carbon-carbonbonds in a sugar ring. The exposed terminal groups can then link to NH₂groups on proteins in a Schiff base linkage which is reduced with NaBH₄.(Kitao, T. and Hattori, K., Nature 265, January 6, pp. 81-82 (1977).)The use of periodate oxidation to conjugate glucan to an mAb isillustrated in Example 5.

f) N-hydroxysuccinimide (NHS) activates a terminal COOH group, forexample, of a peptide, to form an active ester derivative that can becovalently coupled to the protein of the monoclonal antibody. Thismethod has been used to attach 30 molecules of chlorambucil/antibodywith little loss of binding activity. (Smyth, M. et al., J. Natl. CancerInst. 76(3): 503-510 (1986).) The use of the NHS procedure to conjugatemastoparan to an mAb is illustrated in Example 6.

Genetic Engineering Methods for Construction of Vasoconjugates

As an alternative method to the chemical linkage of vasoactive agents tomAb, the genetic sequence of the vasoactive peptides can be engineeredinto the sequence of the mAb as illustrated in Example 11.

Use of Vasoactive Immunoconjugates

Before they arc applied in vivo, the immunoconjugates are evaluated invitro by the proliferation radioimmunoassay described by Bindon et al.,Br. J. Cancer 47: 123-133 (1983), and illustrated in Example 8, todetermine the degree of immunoreactivity and biological activityretained by the product. Only immunoconjugates found to have greaterthan 80% immunoreactivity as compared to the unconjugated antibody areused for in vivo experiments.

A successful immunoconjugate will maximize the clinical effectiveness ofmonoclonal antibody-based diagnosis and therapy. Clinically, thevasoactive immunoconjugate is given before or with the immunodiagnostic,chemotherapeutic, or immunotherapeutic dose so that the tumorvasculature will be made more susceptible to penetration by theeffective agents therein. The time required to produce the maximumvasoactive effect depends on the specific conjugate chosen and itsmechanism of action. It is anticipated that if given before the mAbdoses the minimum time between administration of the vasoactiveimmunoconjugate and the administration of the diagnostic or therapeuticagent is at least about 20 minutes, and the maximum time is about 72hours. The optimal interval between the time of administering thevasoactive immunoconjugate and the dose can be experimentally determinedby animal studies or appropriate studies of the tumor host using labeledimmunoconjugate with the imaging, biodistribution studies, and thehistological methods described above.

The dose of vasoactive immunoconjugate to be given is based on criteriaof medical judgment and experience, both objective and subjective.However, an adequate measure of an effective dose is that amountrequired to increase localization of a subsequently administereddiagnostic or therapeutic agent to an extent which improves the clinicalefficacy of therapy, or accuracy of diagnosis, to a statisticallysignificant degree. Comparison is made between treated and untreatedtumor host animals to whom equivalent doses of the diagnostic ortherapeutic agent are administered. Where applicable, for example in theuse of diagnostic or therapeutic agents that are toxic to normal tissue,an effective dose of vasoactive conjugate is also that which similarlyreduces such toxic effects.

The immunodiagnostic dose may comprise mAb having a specificity for atumor and having a label which is detectable in vivo. In a preferredembodiment, this label comprises a radioactive isotope. Theimmunotherapeutic dose may similarly comprise a clinically useful mAb.This mAb may further be attached to a tumoricidal agent, for example, aradioisotope, a chemotherapeutic drug or a toxin.

EXAMPLE 1 Immunoreactivity of Radiolabeled Monoclonal Antibodies

Raji cells are washed twice in cold PBS containing 1 mg/ml bovine serumalbumin and 0.02% sodium azide. (See, e.g., J. Nat'l Cancer Inst. 37:547-559 (1966) for a description of Raji cells and methods of obtainingsame.) Cells (5×10⁵) resuspended in 100 μ.l of wash buffer are pipettedinto microtiter wells (Immulon Removawell Strips; Dynatech Labs., Inc.,Alexandria, Va.). The microtiter plates are pretreated the previousnight with BSA 10 mg/ml) in PBS with azide in order to prevent theantibody solutions from binding to the wells. (Commercially availablemAbs specific for lymphoma cells, Lym-1 and Lym-2, are available fromTechniclone International, Inc., Tustin, Calif.). Radiolabeled Lym-1 andLym-2 are then added (100,000 cpm/well) in a volume of 100 μ.l/well andthe plates are incubated for 30 minutes at room temperature withconstant shaking. The plates are then washed 4 times by spinning at1,000 rpm for 5 minutes, and aspirating the supernatants with a 12-tipmicromatic manifold, and then resuspending the cells in 200 μ.l of washbuffer using a Titertek Multichannel pipet (Flow Labs, McLean, Va.). Thewells are then separated mechanically and counted in a gamma counter toquantitate the amount of label binding to the cells.

EXAMPLE 2 Conjugation of Physalaemin to Monoclonal Antibodies by the CDIMethod

Physalaemin (Sigma Chemical Co., St. Louis, Mo.) is conjugated tomonoclonal antibodies Lym-1 and Lym-2 by the carbodiimide method.Physalaemin, Lym-1 or Lym-2, and 1-cyclohexyl-3-(2-morpholinoethyl 1)carbodiimide metho-p-toluene sulfonate (CDI) (Aldrich Chemical Co.,Milwaukee, Wis.) are mixed in a 1:3.6:36 ratio by weight and incubatedfor 20 min at pH 5.0 at room temperature. The reaction is terminated bydialysis against PBS, pH 7.2 overnight. The conjugate is purified byFPLC Superose (Pharmacia, Piscataway, N.J.) column chromatography andstored at 4° C. in PBS.

EXAMPLE 3 Conjugation of C3a Peptide to F(ab′), ₂ Monoclonal Antibodiesby the SMCC Method

C3a (57-77) peptide is coupled to F(ab′)₂ monoclonal antibodies using abifunctional reagent, succinimidyl-4-(N-maleimido methyl) cyclohexane1-carboxylate (SMCC). (M. Herman, et al., “Antipeptide antibody ofpredetermined specificity recognize and neutralize the bioactivity ofthe pan-specific hematopoietic IL-3,” J. Immunol. 138: 1099-1104,(1987).)

Monoclonal antibodies are cleaved to F(ab′)₂ fragments using pepsin soas to avoid non-specific binding to leukocytes by the Fc portion of theantibody. C3a (57-77), containing an N-terminal cysteine residue, issynthesized using automated protein synthesis. The C3a peptide (1 mg) isdissolved in 300 μ.l of 4M guanadinium-PBS pH 7.5. The pH is adjusted bydialysis to between 3 and 4 with approximately 3 L of 17% H₃PO₄. Thissolution is placed in a receiving tube (17×100 mm).

The F(ab′)₂ antibody (60 nmole) is dissolved in 1.0 ml PBS, pH 7.5, andreacted with 2400 nmoles “reagent” (SMCC) dissolved in dimethylformamide (DMF) and stirred for 30 minutes at room temperature. TheF(ab′)₂ mixture is applied to a Sephadex (Pharmacia, Piscataway, N.J.)G-10 column (2 ml), centrifuged at 1500 G for 1 minute and collected inthe receiving tube. The column is washed with 300 μ.l PBS, pH 7.5, andre-centrifuged. The pH is adjusted to between 7.0 and 7.7 and themixture is stirred for 3 hours at room temperature.

The conjugated antibody is stored at 4° C.

C5a is coupled to F(ab′)₂ antibody using the bifunctional cross-linkingreagents dimethyl superimidate or SPDP. Conditions will be adjusted toproduce 1/1 C5a-F(ab)₂ conjugates and to minimize polymerization ofeither C5a or F(ab)₂ alone.

EXAMPLE 4 Conjugation of Viprostol to Monoclonal Antibodies byCis-Aconityl Derivatization

Viprostol can be derivatized by adding a cis-aconityl spacer arm throughits 11-hydroxy group. In this reaction, Viprostol (5 mg) is dissolved in1 ml of 0.1M Na₂ HPO₄ in a test tube and cooled in an ice bath. A molarexcess (5 mg) of cis-aconityl anhydride (Aldrich Chemicals, Milwaukee,Wis.) is added slowly to the solution while stirring, and the pH is keptbetween 8 and 9 by careful addition of 1N NaOH. Thin-layerchromatography of samples is used to monitor the progress of thereaction. A tracer of ³H- or ¹⁴C-labeled Viprostol may be added to thereaction mixture and the progress of the reaction monitored byautoradiography of the thin-layer plates. Separation and purification ofthe derivative may be achieved by using acidification and purificationof the product or a column chromatography method. To conjugate Viprostolto monoclonal antibodies, a solution of the cis-aconityl Viprostolderivative is added to a solution containing Lym-1 or Lym-2 monoclonalantibodies. The mixture is then allowed to incubate for 30 minutes atroom temperature at pH 5.0. The reaction is terminated and the conjugatepurified by eluting the sample through a Sephadex G-25 column, or byFPLC Superose column chromatography.

EXAMPLE 5 Conjugation of Glucan to Monoclonal Antibodies by PeriodateOxidation

Glucan is carefully oxidized by using periodate oxidation to cleave oneof its sugar moieties without affecting its bioactivity, and using onlya 1 to 2 molar excess of NaIO₄. The aldehyde groups produced in theglucan by oxidation will react with —NH₂ groups on the monoclonalantibodies to form a Schiff base. The Schiff base linkage is thenreduced with NaBH₄, at a concentration of 0.3 mg/ml to form a stableamine linkage of glucan-Mab conjugate.

EXAMPLE 6 Conjugation of Mastoparan to Monoclonal Antibodies by the NHSMethod

An active ester of mastoparan is prepared by reaction withN-hydroxysuccinimide (NHS) in dimethylformamide and usingN,N-dicyclohexylcarbodiimide (DCC) as a condensation reagent. A solutionof the mastoparan active ester in DMF is then added to a solutioncontaining Lym-1 or Lym-2 monoclonal antibodies at pH 7.0 and allowed toreact for 1 to 2 hours at room temperature. Any undissolved reagents,mainly dicyclohexyl urea and/or precipitated protein, are removed bycentrifugation. Free mastoparan and other unreacted starting materialscan be removed by gel filtration chromatography using a Sephadex G-25(Pharmacia, Piscataway, N.J.) column. The amount of mastoparanincorporated in the conjugate is determined by means of a tracer oftritium (H³)-labelled mastoparan.

EXAMPLE 7 Conjugation of Interleukin-2 to Tumor-Specific MonoclonalAntibodies by CDI

Recombinant Interleukin-2 (rIL-2) (Cetus Corporation, Emeryville,Calif.) is provided in vials containing 0.3 mg or 1.2 mg/vial. Purifiedmonoclonal antibody such as Lym-1 is conjugated to rIL-2 using1-cyclohexyl-3-(2-morpholinoethyl carbodiimide metho-p-toluenesulfonate)(“CDI”) and N-hydroxysulfosuccinimide in a 1:2:50:50 ratio by weight togive a total volume of 0.3 ml in phosphate buffer, pH 7.4. The reactionmixture is incubated overnight at 4° C. After centrifugation at 4000 rpmfor 15 minutes at 4° C., the soluble coupled antibody is chromatographedon a Sephadex G-100 column calibrated with blue dextran. Using thisprocedure, approximately 1-2 molecules of rIL-2 are coupled to eachmonoclonal antibody (“mAb”) molecule. The immunoconjugate preparation isthen adjusted to 1 mg/ml, sterile filtered, and stored at 4° C. untiluse. This procedure can be used to couple rIL-2 to any tumor-specificmonoclonal antibody.

EXAMPLE 8 Conjugation of Interleukin-2 to a Therapeutic Agent

The same conjugation procedure described above can be used to link rIL-2to a monoclonal antibody with specificity for tumor endothelium. As anexample, monoclonal antibodies to fibronectin have been shown to bindselectively to tumor vasculature compared to normal endothelium. Sincethe primary action of the rIL-2 at the tumor site is to enhance vascularpermeability, targeting the vasoactive immunoconjugate to the tumorendothelium would be optimal. In addition, using a similar methodology,rIL-2 conjugate treatment may then be supplemented or followed by use ofconventional chemotherapeutic agents including, for example,cis-platinum, for the treatment of different types of cancers.

EXAMPLE 9 Recombinantly Engineered Vasoactive Immunoconjugate

Instead of chemically linking vasoactive peptides to monoclonalantibodies which target tumors or tumor vasculature, the geneticsequence of the vasoactive peptide can be engineered into the sequenceof the monoclonal antibody. As an example, mRNA coding for theanti-fibronectin monoclonal antibody is isolated. From this mRNA, a cDNAis synthesized for both the heavy and light chains of immunoglobulin.This cDNA is subsequently 1) amplified using the polymerase chainreaction; 2) sequenced; and 3) mapped by restriction endonucleases. Theappropriate DNA sequence of the vasoactive peptide, such as IL-2, isthen ligated to the ends of the heavy chain gene in the constant region.

The completed engineered gene is then reintroduced into a eukaryotic orprokaryotic expression system by gene transfection methods (for example,using electroporation or the calcium phosphate method), so that theprotein product is expressed in large scale cell culture. As illustratedin FIG. 1, two active IL-2 moieties will be part of each immunoglobulinmolecule. The best site of attachment for each vasoactive peptide may bedifferent and may easily be determined via experimental methods. Thesequence for the vasoactive peptide can be ligated to either human ormouse immunoglobulin heavy chain sequences to produce human, mouse,chimeric or other species or combinations of immunoglobulin molecules.

EXAMPLE 10 Functional Activity of Immunoconjugates as Determined by aProliferation Assay

In order to test the functional activity of the rIL-2 immunoconjugate, aproliferation assay is performed.

Fresh murine splenocytes are placed in culture with rIL-2 for 7 daysafter stimulation by PHA for 3 days. The cells are then washed free ofrIL-2 and resuspended in RPMI-1640 medium supplemented with 10% fetalcalf serum and antibiotics. One hundred μ.l containing 10⁵ cells areplaced in triplicate in microtiter plate wells in the presence of 100μ.l of varying concentrations of immunoconjugate (test sample), rIL-2(positive control) and Lym-1 or Lym-2 (negative control). Cultures areincubated for 24 hours at 37° C. after which time 2 μ.Ci of ¹²⁵I-IUDR(New England Nuclear Co., Boston, Mass.) are added for a 4 hourincubation period. Cells are then harvested by washing 3 times with PBSand once with 5% trichloracetic acid before being counted in a gammacounter. Using purified rIL-2 as a positive control, the ¹²⁵I-IUDRincorporation data can be plotted against log₂ of rIL-2 dilution togenerate a dose response curve. The x-axis dilution coordinate of thecontrol sample which crosses this curve at the 50% maximum ¹²⁵I-IUDRuptake (y-axis coordinate) is defined at that value which corresponds to1 unit of rIL-2 activity. In this way, the rIL-2 activity of theimmunoconjugate preparations can be quantitated from batch to batch.

EXAMPLE 11 Use of Vasoactive Compounds to Increase Extravascular TumorPenetration by Monoclonal Antibody Lym-1

To test the relative effects of the IL-2 vasoactive immunoconjugate onthe biodistribution and tumor uptake of Lym-1 in lymphoma-bearing nudemice, groups of five mice each bearing 0.5 g Raji lymphoma subcutaneoustransplants were given intravenous doses of Lym-1 (control) orLym-1/IL-2 immunoconjugate (experimental) at times zero or 2½ hoursbefore the administration of 20 μ.g of Lym-1 F(ab′)₂ radiolabeled with50 μ.Ci of I-125. Three days later, all the mice were sacrificed and thetumors and normal organs removed to quantitate the amount of label pergram of tissue. As shown below, those mice receiving the experimentalLym-1/IL-2 vasoconjugate showed a 200% increase in mAb localization overappropriate controls (FIG. 1). Furthermore, as illustrated in thefollowing FIGS. 2-4, this increase in mAb localization enhances thetumor/blood ratio approximately twofold (FIG. 2), is dose-dependent(maximum effect between 30-50 μ.g of vasoconjugate; FIG. 3), and is timedependent (FIG. 4) with a maximum effect demonstrated 2½ hours beforethe administration of the mAb.

EXAMPLE 12 Clinical Use and Application

It is intended that vasoactive immunoconjugates be used to enhance thedelivery of 1) drugs or drug-containing liposomes, and 2) therapeuticmonoclonal antibodies. The mechanism of action of the immunoconjugate isthe production of an increase in the permeability and/or blood flow atthe tumor site. Hence, the immunoconjugate is generally administered 1-3hours before the therapeutic dose of drug, monoclonal antibody, orliposome is administered.

In an animal model, the use of rIL-2 linked to Lym-1 2½ hours before theadministration of I-131 Lym-1 F(ab′)₂ increases the dose of the latterby 200% compared to controls. The use of engineered immunoconjugates maysignificantly increase their effectiveness in vivo.

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentis to be considered in all respects only as illustrative and notrestrictive and the scope of the invention is, therefore, indicated bythe appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. A pharmaceutical conjugate, comprising: anantibody which localizes at the site of neoplastic tissue in a mammal;and a chemical agent that increases the blood supply to said neoplastictissue, provided that said agent is not tumor necrosis factor,formyl-methionyl-leucyl-phenylalanine (fMLP), nor cobra venom factor. 2.The conjugate of claim 1, wherein said conjugate is of sufficient sizeto be unable to penetrate normal, healthy vascular endothelium, but ableto penetrate the vascular endothelium of tumor tissue.
 3. The conjugateof claim 1, wherein said agent increases vasopermeability at an activesite in vascular endothelium.
 4. The conjugate of claim 1, wherein saidagent provokes or exacerbates a local inflammatory reaction at an activesite in vascular endothelium.
 5. The conjugate of claim 1, wherein saidconjugate comprises a radioisotope.
 6. The conjugate of claim 1, whereinsaid agent comprises a pharmaceutically active compound.
 7. Theconjugate of claim 1, wherein said agent is a biological amine.
 8. Theconjugate of claim 1, wherein said antibody has specificity formolecules that are selectively expressed in vascular endothelium that isdamaged, inflamed or structurally abnormal.
 9. The conjugate of claim 1,wherein said antibody comprises monoclonal antibody.
 10. The conjugateof claim 1, wherein said antibody is an antibody fragment selected fromthe group consisting of Fab, HL and F(ab′)₂ fragments.
 11. The conjugateof claim 1, wherein said antibody has specificity for subendothelialepitopes of the blood vessel wall that are accessible to circulatingantibody or other macromolecules in inflamed vessels and in structurallyabnormal vessels such as those found in tumors.
 12. The conjugate ofclaim 11, wherein said epitopes are found in components selected fromthe group consisting of fibronectin, laminin, and type IV collagen. 13.The conjugate of claim 1, wherein said antibody has specificity forantigens selectively expressed in endothelial cells in inflamed vasculartissue, but not in non-inflamed vascular tissue.
 14. The conjugate ofclaim 13, wherein said antigens include cell adhesion moleculesresponsible for adherence of polymorphonuclear leukocytes to inflamedvascular tissue.
 15. The conjugate of claim 13, wherein said antigenscomprise fibronectin.
 16. The conjugate of claim 1, wherein saidantibody has specificity for antigens selectively expressed inendothelial cells in new vascular tissue.
 17. A method for treatment ofneoplastic tissue, comprising: administering to a host having saidtissue the pharmaceutical conjugate of claim 1; and contemporaneously orthereafter administering to said host an antineoplastic agent.
 18. Amethod according to claim 17, wherein said antineoplastic agent isadministered as a conjugate, said conjugate comprising an antibodyhaving the ability to localize at the site of said tissue, conjugated tosaid antineoplastic agent.